Tamoxifen activity against Plasmodium in vitro and in mice

Abstract Background Tamoxifen is an oestrogen receptor modulator that is widely used for the treatment of early stage breast cancer and reduction of recurrences. Tamoxifen is also used as a powerful research tool for controlling gene expression in the context of the Cre/loxP site-specific recombination system in conditional mutant mice. Methods To determine whether the administration of tamoxifen affects Plasmodium growth and/or disease outcome in malaria, in vitro studies assessing the effect of tamoxifen and its active metabolite 4-hydroxytamoxifen on Plasmodium falciparum blood stages were performed. Tamoxifen effects were also evaluated in vivo treating C57/B6 mice infected with Plasmodium berghei (ANKA strain), which is the standard animal model for the study of cerebral malaria. Results Tamoxifen and its active metabolite, 4-hydroxytamoxifen, show activity in vitro against P. falciparum (16.7 to 5.8 µM IC50, respectively). This activity was also confirmed in tamoxifen-treated mice infected with P. berghei, which show lower levels of parasitaemia and do not develop signs of cerebral malaria, compared to control mice. Mice treated with tamoxifen for 1 week and left untreated for an additional week before infection showed similar parasitaemia levels and signs of cerebral malaria as control untreated mice. Conclusions Tamoxifen and its active metabolite, 4-hydroxytamoxifen, have significant activity against the human parasite P. falciparum in vitro and the rodent parasite P. berghei in vivo. This activity may be useful for prevention of malaria in patients taking this drug chronically, but also represents a major problem for scientists using the conditional mutagenic Cre/LoxP system in the setting of rodent malaria. Allowing mice to clear tamoxifen before starting a Plasmodium infection allows the use the Cre/LoxP conditional mutagenic system to investigate gene function in specific tissues.

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In Silico Predicted Antifungal Peptides: In Vitro and In Vivo Anti-Candida Activity

Journal of Fungi ◽

10.3390/jof7060439 ◽

2021 ◽

Vol 7 (6) ◽

pp. 439

Author(s):

Tecla Ciociola ◽

Walter Magliani ◽

Tiziano De Simone ◽

Thelma A. Pertinhez ◽

Stefania Conti ◽

...

Keyword(s):

In Silico ◽

Mammalian Cells ◽

Galleria Mellonella ◽

Ex Vivo ◽

Consensus Sequence ◽

In Silico Analysis ◽

Significant Activity ◽

Synthetic Antibody

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

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Antitrypanosomal activity of purine nucleosides can be enhanced by their conversion to O-acetylated derivatives.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.11.2567 ◽

1996 ◽

Vol 40 (11) ◽

pp. 2567-2572 ◽

Cited By ~ 11

Author(s):

J R Sufrin ◽

D Rattendi ◽

A J Spiess ◽

S Lane ◽

C J Marasco ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Parent Compound ◽

Nucleoside Analogs ◽

Significant Activity ◽

Structural Class ◽

Antitrypanosomal Activity ◽

Salvage Pathways ◽

Purine Salvage Pathways

Fifteen purine nucleosides and their O-acetylated ester derivatives were examined for in vitro antitrypanosomal activity against the LAB 110 EATRO isolate of Trypanosoma brucei brucei and two clinical isolates of Trypanosoma brucei rhodesiense. Initial comparisons of activity were made for the LAB 110 EATRO isolate. Three nucleoside analogs exhibited no significant activity (50% inhibitory concentrations [IC50s] of > 100 microM), whether they were O acetylated or unacetylated; three nucleosides showed almost equal activity (IC50s of < 5 microM) for the parent compound and the O-acetylated derivative; nine nucleosides showed significantly improved activity (> or = 3-fold) upon O acetylation; of these nine analogs, six displayed activity at least 10-fold greater than that of their parent nucleosides. The most significant results were those for four apparently inactive compounds which, upon O acetylation, displayed IC50s of < or = 25 microM. When the series of compounds was tested against T. brucei rhodesiense isolates (KETRI 243 and KETRI 269), their antitrypanosomal effects were comparable to those observed for the EATRO 110 strain. Thus, our studies of purine nucleosides have determined that O acetylation consistently improved their in vitro antitrypanosomal activity. This observed phenomenon was independent of their cellular enzyme targets (i.e., S-adenosylmethionine, polyamine, or purine salvage pathways). On the basis of our results, the routine preparation of O-acetylated purine nucleosides for in vitro screening of antitrypanosomal activity is recommended, since O acetylation transformed several inactive nucleosides into compounds with significant activity, presumably by improving uptake characteristics. O-acetylated purine nucleosides may offer in vivo therapeutic advantages compared with their parent nucleosides, and this possibility should be considered in future evaluations of this structural class of trypanocides.

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THEMIS Is Required for Pathogenesis of Cerebral Malaria and Protection against Pulmonary Tuberculosis

Infection and Immunity ◽

10.1128/iai.02586-14 ◽

2014 ◽

Vol 83 (2) ◽

pp. 759-768 ◽

Cited By ~ 9

Author(s):

Sabrina Torre ◽

Sebastien P. Faucher ◽

Nassima Fodil ◽

Silayuv E. Bongfen ◽

Joanne Berghout ◽

...

Keyword(s):

Pulmonary Tuberculosis ◽

Cerebral Malaria ◽

Functional Coupling ◽

Experimental Cerebral Malaria ◽

Content Type ◽

Proinflammatory Responses ◽

Inflammatory Bowel ◽

Blood Brain Barrier Integrity

We identify anN-ethyl-N-nitrosourea (ENU)-induced I23N mutation in the THEMIS protein that causes protection against experimental cerebral malaria (ECM) caused by infection withPlasmodium bergheiANKA.ThemisI23Nhomozygous mice show reduced CD4+and CD8+T lymphocyte numbers. ECM resistance inP. bergheiANKA-infectedThemisI23Nmice is associated with decreased cerebral cellular infiltration, retention of blood-brain barrier integrity, and reduced proinflammatory cytokine production. THEMISI23Nprotein expression is absent from mutant mice, concurrent with the decreased THEMISI23Nstability observedin vitro. Biochemical studiesin vitroand functional complementationin vivoinThemisI23N/+:Lck−/+doubly heterozygous mice demonstrate that functional coupling of THEMIS to LCK tyrosine kinase is required for ECM pathogenesis. Damping of proinflammatory responses inThemisI23Nmice causes susceptibility to pulmonary tuberculosis. Thus, THEMIS is required for the development and ultimately the function of proinflammatory T cells.ThemisI23Nmice can be used to study the newly discovered association ofTHEMIS(6p22.33) with inflammatory bowel disease and multiple sclerosis.

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Upregulations of Clcn3 and P-Gp Provoked by Lens Osmotic Expansion in Rat Galactosemic Cataract

Journal of Diabetes Research ◽

10.1155/2017/3472735 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8

Author(s):

Lixia Ji ◽

Lixia Cheng ◽

Zhihong Yang

Keyword(s):

Osmotic Stress ◽

Early Stage ◽

Lens Epithelial Cells ◽

Anterior Capsule ◽

Glycoprotein P ◽

Protein Levels ◽

P Glycoprotein ◽

Sugar Cataract

Objective.Lens osmotic expansion, provoked by overactivated aldose reductase (AR), is the most essential event of sugar cataract. Chloride channel 3 (Clcn3) is a volume-sensitive channel, mainly participating in the regulation of cell fundamental volume, and P-glycoprotein (P-gp) acts as its modulator. We aim to study whether P-gp and Clcn3 are involved in lens osmotic expansion of galactosemic cataract.Methods and Results.In vitro, lens epithelial cells (LECs) were primarily cultured in gradient galactose medium (10–60 mM), more and more vacuoles appeared in LEC cytoplasm, and mRNA and protein levels of AR, P-gp, and Clcn3 were synchronously upregulated along with the increase of galactose concentration. In vivo, we focused on the early stage of rat galactosemic cataract, amount of vacuoles arose from equatorial area and scattered to the whole anterior capsule of lenses from the 3rd day to the 9th day, and mRNA and protein levels of P-gp and Clcn3 reached the peak around the 9th or 12th day.Conclusion. Galactosemia caused the osmotic stress in lenses; it also markedly leads to the upregulations of AR, P-gp, and Clcn3 in LECs, together resulting in obvious osmotic expansion in vitro and in vivo.

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Rationally Designing Simple Rod-Like Amphiphilic NIR-Emissive AIE Probes for Precise In-Vivo Detection of Aβ Fibrils/Plaques at A Super-Early Stage

10.26434/chemrxiv.13699222.v1 ◽

2021 ◽

Author(s):

Yipu Wang ◽

Dong Mei ◽

Xinyi Zhang ◽

Da-Hui Qu ◽

Ju Mei ◽

...

Keyword(s):

High Performance ◽

Ex Vivo ◽

Early Stage ◽

Signal To Noise Ratio ◽

High Signal ◽

In Vivo Detection ◽

Different Strains ◽

Aβ Fibrils

With increase of social aging, Alzheimer's disease (AD) has been one of the serious diseases threatening human health. The occurrence of Aβ fibrils or plaques is recognized as the hallmark of AD. Currently, optical imaging has stood out to be a promising technique for the imaging of Aβ fibrils/plaques and the diagnosis of AD. However, restricted by their poor blood-brain barrier (BBB) penetrability, short-wavelength excitation and emission, and aggregation-caused quenching (ACQ) effect, the clinically used gold-standard optical probes such as <a>thioflavin</a> T (ThT) and thioflavin S (ThS), are not effective enough in the early diagnosis of AD in vivo. Herein, we put forward an “all-in-one” design principle and demonstrate its feasibility in developing high-performance fluorescent probes which are specific to Aβ fibrils/plaques and promising for super-early in-vivo diagnosis of AD. As a proof of concept, a simple rod-like amphiphilic NIR fluorescent AIEgen, i.e., AIE-CNPy-AD, is developed by taking the specificity, BBB penetration ability, deep-tissue penetration capacity, high signal-to-noise ratio (SNR) into consideration. AIE-CNPy-AD is constituted by connecting the electron-donating and accepting moieties through single bonds and tagging with a propanesulfonate tail, giving rise to the NIR fluorescence, aggregation-induced emission (AIE) effect, amphiphilicity, and rod-like structure, which in turn result in high binding-affinity and excellent specificity to Aβ fibrils/plaques, satisfactory ability to penetrate BBB and deep tissues, ultrahigh SNR and sensitivity, and high-fidelity imaging capability. In-vitro, ex-vivo, and in-vivo <a>identifying of Aβ fibrils/plaques</a> in different strains of mice indicate that AIE-CNPy-AD holds the universality to the detection of Aβ fibrils/plaques. It is noteworthy that AIE-CNPy-AD is even able to trace the small and sparsely distributed Aβ fibrils/plaques in very young AD model mice such as 4-month-old APP/PS1 mice which are reported to be the youngest mice to have Aβ deposits in brains, suggesting its great potential in diagnosis and intervention of AD at a super-early stage.

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Defining the early stages of intestinal colonisation by whipworms

10.1101/2020.08.21.261586 ◽

2020 ◽

Author(s):

María A. Duque-Correa ◽

David Goulding ◽

Claire Cormie ◽

Catherine Sharpe ◽

Judit Gali Moya ◽

...

Keyword(s):

Intestinal Epithelium ◽

Early Stage ◽

Proximal Colon ◽

Host Cells ◽

Parasitic Nematodes ◽

Metazoan Parasites ◽

Multiple Cells ◽

First Time

ABSTRACTHundreds of millions of people are infected with whipworms (Trichuris trichiura), large metazoan parasites that live in the caecum and proximal colon. Whipworms inhabit distinct multi-intracellular epithelial burrows that have been described as syncytial tunnels. However, the interactions between first-stage (L1) larvae and the host epithelia that determine parasite invasion and establishment in the syncytium remain unclear. In vivo experiments investigating these events have been severely hampered by the limited in situ accessibility to intracellular infective larvae at the bottom of the crypts of Lieberkühn, and the lack of genetic tools such as fluorescent organisms that are readily available for other pathogens but not parasitic nematodes. Moreover, cell lines, which do not mimic the complexity of the intestinal epithelium, have been unsuccessful in supporting infection by whipworm larvae. Here, we show that caecaloids grown in an open crypt-like conformation recapitulate the caecal epithelium. Using this system, we establish in vitro infections with T. muris L1 larvae for the first-time, and provide clear evidence that syncytial tunnels are formed at this early stage. We show that larval whipworms are completely intracellular but woven through multiple cells. Using the caecaloids, we are able to visualise the pathways taken by the larvae as they burrow through the epithelial cells. We also demonstrate that larvae degrade the mucus layers overlaying the epithelium, enabling them to access the cells below. We show that early syncytial tunnels are composed of enterocytes and goblet cells that are alive and actively interacting with the larvae during the first 24 h of the infection. Progression of infection results in damage to host cells and by 72 h post-infection, we show that desmosomes of cells from infected epithelium widen and some host cells appear to become liquified. Collectively, our work unravels processes mediating the intestinal epithelium invasion by whipworms and reveals new specific interactions between the host and the parasite that allow the whipworm to establish on its multi-intracellular niche. Our study demonstrates that caecaloids can be used as a relevant in vitro model to investigate the infection biology of T. muris during the early colonisation of its host.

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TArget-Responsive Subcellular Catabolism Analysis for Early-stage Antibody-Drug Conjugates Screening and Assessment

10.26434/chemrxiv.13309454 ◽

2020 ◽

Author(s):

Hua Sang ◽

Jiali Liu ◽

Fang Zhou ◽

Xiaofang Zhang ◽

Jingwei Zhang ◽

...

Keyword(s):

Quality Assessment ◽

Therapeutic Efficacy ◽

High Throughput Screening ◽

Early Stage ◽

Cellular Level ◽

Drug Conjugates ◽

Therapeutic Outcomes ◽

Lysosomal Proteolysis

Key events including antibody-antigen affinity, ADC internalization, trafficking and lysosomal proteolysis-mediated payload release combinatorially determine the therapeutic efficacy and safety for ADCs. Nevertheless, a universal technology that efficiently and conveniently evaluates the involvement of these above elements to ADC payload release and hence the final therapeutic outcomes for mechanistic studies and quality assessment is lacking. Considering the plethora of ADC candidates under development owing to the ever-evolving linker and drug chemistry, we developed a TArget-Responsive Subcellular Catabolism (TARSC) approach that measures catabolites kinetics for given ADCs and elaborates how each individual step ranging from antigen binding to lysosomal proteolysis affects ADC catabolism by targeted interferences. Using a commercial and a biosimilar ado-trastuzumab emtansine (T-DM1) as model ADCs, we recorded unequivocal catabolites kinetics for the two T-DM1s in the presence and absence of the targeted interferences. Their negligible differences in TARSC profiles fitting with their undifferentiated therapeutic outcomes suggested by in vitro viability assays and in vivo tumor growth assays, highlighting TARSC analysis as a good indicator of ADC efficacy and bioequivalency. Lastly, we demonstrated the use of TARSC in assessing payload release efficiency for a new Trastuzumab-toxin conjugate. Collectively, we demonstrated the use of TARSC in characterizing ADC catabolism at (sub)cellular level, and in systematically depicting whether given target proteins affect ADC payload release and hence therapeutic efficacy. We anticipate its future use in high-throughput screening, quality assessment and mechanistic understanding of ADCs for drug R&D before proceeding to costly in vivo experiments.

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Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos

10.21203/rs.3.rs-73275/v2 ◽

2020 ◽

Author(s):

Vera A van der Weijden ◽

Meret Schmidhauser ◽

Mayuko Kurome ◽

Johannes Knubben ◽

Veronika L Flöter ◽

...

Keyword(s):

Signaling Pathways ◽

Early Stage ◽

Developmental Competence ◽

Molecular Signaling ◽

Developmental Potential ◽

Stage Of Development ◽

Molecular Signaling Pathways ◽

Canonical Pathways

Abstract Background: The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts.Results: In vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation, tRNA charging, and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14-3-3-mediated signaling, signaling of Rho family GTPases, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts.Conclusions: Our findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.

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CSPα reduces aggregates and rescues striatal dopamine release in αsynuclein transgenic mice

10.1101/2020.07.31.229153 ◽

2020 ◽

Author(s):

L Caló ◽

E Hidari ◽

M Wegrzynowicz ◽

JW Dalley ◽

BL Schneider ◽

...

Keyword(s):

Dopamine Release ◽

Early Stage ◽

Synaptic Function ◽

Snare Complex ◽

Early Event ◽

Vesicle Recycling ◽

Cysteine String Protein ◽

And Function

AbstractαSynuclein aggregation at the synapse is an early event in Parkinson’s disease and is associated with impaired striatal synaptic function and dopaminergic neuronal death. The cysteine string protein (CSPα) and αsynuclein have partially overlapping roles in maintaining synaptic function and mutations in each cause neurodegenerative diseases. CSPα is a member of the DNAJ/HSP40 family of co-chaperones and like αsynuclein, chaperones the SNARE complex assembly and neurotransmitter release. αSynuclein can rescue neurodegeneration in CSPαKO mice. However, whether αsynuclein aggregation alters CSPα expression and function is unknown. Here we show that αsynuclein aggregation at the synapse induces a decrease in synaptic CSPα and a reduction in the complexes that CSPα forms with HSC70 and STGa. We further show that viral delivery of CSPα rescues in vitro the impaired vesicle recycling in PC12 cells with αsynuclein aggregates and in vivo reduces synaptic αsynuclein aggregates restoring normal dopamine release in 1-120hαsyn mice. These novel findings reveal a mechanism by which αsynuclein aggregation alters CSPα at the synapse, and show that CSPα rescues αsynuclein aggregation-related phenotype in 1-120hαsyn mice similar to the effect of αsynuclein in CSPαKO mice. These results implicate CSPα as a potential therapeutic target for the treatment of early-stage PD.

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A Novel Piperazine-Based Drug Lead for Cryptosporidiosis from the Medicines for Malaria Venture Open-Access Malaria Box

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01505-17 ◽

2018 ◽

Vol 62 (4) ◽

pp. e01505-17 ◽

Cited By ~ 17

Author(s):

R. S. Jumani ◽

K. Bessoff ◽

M. S. Love ◽

P. Miller ◽

E. E. Stebbins ◽

...

Keyword(s):

Mouse Model ◽

Drug Development ◽

Early Stage ◽

New Drugs ◽

Cryptosporidium Hominis ◽

Content Type ◽

Knockout Mouse Model ◽

Malaria Box

ABSTRACTCryptosporidiosis causes life-threatening diarrhea in children under the age of 5 years and prolonged diarrhea in immunodeficient people, especially AIDS patients. The standard of care, nitazoxanide, is modestly effective in children and ineffective in immunocompromised individuals. In addition to the need for new drugs, better knowledge of drug properties that drivein vivoefficacy is needed to facilitate drug development. We report the identification of a piperazine-based lead compound forCryptosporidiumdrug development, MMV665917, and a new pharmacodynamic method used for its characterization. The identification of MMV665917 from the Medicines for Malaria Venture Malaria Box was followed by dose-response studies,in vitrotoxicity studies, and structure-activity relationship studies using commercial analogues. The potency of this compound againstCryptosporidium parvumIowa and field isolates was comparable to that againstCryptosporidium hominis. Furthermore, unlike nitazoxanide, clofazimine, and paromomycin, MMV665917 appeared to be curative in a NOD SCID gamma mouse model of chronic cryptosporidiosis. MMV665917 was also efficacious in a gamma interferon knockout mouse model of acute cryptosporidiosis. To determine if efficacy in this mouse model of chronic infection might relate to whether compounds are parasiticidal or parasitistatic forC. parvum, we developed a novelin vitroparasite persistence assay. This assay suggested that MMV665917 was parasiticidal, unlike nitazoxanide, clofazimine, and paromomycin. The assay also enabled determination of the concentration of the compound required to maximize the rate of parasite elimination. This time-kill assay can be used to prioritize early-stageCryptosporidiumdrug leads and may aid in planningin vivoefficacy experiments. Collectively, these results identify MMV665917 as a promising lead and establish a new method for characterizing potential anticryptosporidial agents.

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